1 Project Summary 2 3 The 3D topology of the genome plays a critical role in transcriptional regulation in health, disease and 4 development. While techniques such as Hi-C and ChIA-PET have revealed static views of global genome 5 architecture, very little is known about the mechanisms that control dynamic topological rearrangements. We 6 have established a system in budding yeast ? the Hsf1-mediated heat shock response ? in which concerted 7 structural rearrangements within and between a set of specific target genes take place. During heat shock, 8 Hsf1 drives its target genes dispersed on different chromosomes to undergo striking changes in conformation 9 and coalesce into discrete, transcriptionally active foci. These Hsf1 target genes encode a dedicated group of 10 protein homeostasis (proteostasis) factors. Through their regulation, the human orthologue of Hsf1 (hHSF1) 11 has been suggested to be a clinical target in malignant cancers and in neurodegenerative diseases. Thus, this 12 proposal has dual significance: it will both elucidate mechanisms that control chromatin conformational 13 dynamics and reveal how Hsf1 coordinates expression of the proteostasis machinery. 14 The Hsf1-mediated heat shock response in budding yeast represents an ideal model system to investigate 15 the regulation and function of 3D genome rearrangements for two reasons: 1) The magnitude, rapidity and 16 specificity of the intra- and intergenic rearrangements are all unprecedented and thus represent exciting, novel 17 biology; and 2) It will allow us to leverage the power of yeast gene-tics to dissect the mechanisms and define 18 the functional relevance of genome topology dynamics. 19 Aim 1 will investigate the 3D rearrangements that occur genome-wide during heat shock and the role 20 played by Hsf1 and RNA polymerase II (including its CTD phosphorylation state) in orchestrating specific and 21 robust interchromosomal contacts. The experiments will use primarily ChIA-PET approaches. 22 Aim 2 will focus deeply on the role of Hsf1 binding sites and its functional domains in underpinning its 23 ability to dynamically drive members of its regulon into coalesced intranuclear foci. 24 Aim 3 will test the role of Mediator and other cofactors - transcriptional co-activators, chromatin remodelers 25 and architectural proteins - in driving HSP gene coalescence and investigate the possibility that HSP gene 26 coalescence represents a condensate that assembles through liquid-liquid phase separation. 27 In support of the NIH mission, the precedents established in this proposal will inform therapeutic efforts 28 aimed broadly at 3D genome regulation and may suggest novel molecular handles with which to modulate 29 Hsf1 and the proteostasis machinery to treat cancer and neurodegenerative diseases.